A number of bacterial natural products contain substituted a,B-unsaturated-d&#61485;lactones. The chemical nature of the substituents alters the biological activity considerably. For natural products such as fostriecin and phoslactomycins (PLMs), the d-substituent is a linear polyketide chain and unusual in that it has multiple cis double bonds and a phosphate group. The PLMs exhibit remarkably selectivity and potent activity against human fungal pathogens, including Aspergillus fumigatus. The DNA encoding the PLM and fostriecin biosynthetic processes have been cloned, sequenced and analyzed. It has been shown that both natural products are generated by modular polyketide synthases (PKSs). In the case of PLM much of the biosynthetic process has been deciphered. The knowledge and genetic tools provided through ongoing PLM biosynthetic studies has allowed the generation of blocked mutants, hybrid pathways, and mutasynthetic approaches to access new natural products based around the PLM skeleton. These compounds are generally obtained in excellent fermentation yield (10-50 mg/L) from fermentations and include significant structural diversity both around the lactone core and in the delta substituent. Additional structural variation has been obtained by chemical and efficient enzymatic modification of these new PLM core structures. The proposal has 4 aims. Aim 1 will test a hypothesis for how modules in both systems establish the unusual cis double bonds in the d- substituent. Aim 2-3 will test a hypothesis that the cis double bond of the unsaturated lactone, is not established by a canonical dehydration domain within a PKS module, but rather an unprecedented decarboxylative elimination from a malonylated pathway intermediate. A series of chemical, biochemical, genetic and structural approaches will investigate the enzyme-catalyzed elimination reaction, determine how the malonylated intermediate is generated, and the sequence of these steps in the PLM biosynthetic pathway. Aims 4 will focus on evaluating the antifungal activity of new PLMs, and the basis for their selective antifungal activity.